Synthetic lipid-like materials for brain delivery

ABSTRACT

Disclosed are (i) compounds of formula I, or pharmaceutically acceptable salts thereof; and (ii) lipidoid nanoparticles comprising compound of formula I or pharmaceutically acceptable salts thereof, as well as their use as vehicles for drug delivery across the blood-brain barrier.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.17/339,563, filed Jun. 4, 2021; which is a continuation of InternationalPatent Application No. PCT/US2021/030664, filed May 4, 2021; whichclaims the benefit of priority to U.S. Provisional Application No.63/019,530, filed May 4, 2020.

GOVERNMENT SUPPORT

This invention was made with government support under grant numbersTR002636 and EB027170 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

The treatment of central nervous systems (CNS) diseases, such asneurodegenerative disorders, brain tumors, brain infections, and stroke,is severely constrained by the blood-brain-barrier (BBB) because itprevents the transfer of most of small molecule drugs and macromolecules(e.g., peptides, gene drugs, and protein drugs) into the brain. To date,extensive efforts have been undertaken to enhance brain deliveryefficiency, including direct CNS administration, disruption of the BBB,and carrier vehicle mediated delivery. However, direct administration tothe CNS is invasive, which may cause infection and tissue damage, and isalso limited by diffusion distance and rapid efflux of drugs out of theCNS within hours. Disruption of the BBB, using techniques such asosmotic disruption, biochemical disruption, and ultrasound-mediateddisruption, is effective to introduce drugs into brain, however, thesetransient BBB openings also allows for the leakage of plasma proteinsinto the brain, leading to neurotoxicity, vascular pathology, andchronic neuropathologic changes in the brain. Therefore, approaches forsafe and efficient delivery of BBB-impermeable cargos, in particular forgene and nucleic acid therapy, into CNS remain to be desired.

The carrier vehicle mediated brain drug delivery is considered apromising and versatile brain delivery system. For decades, variouscarrier vehicles, such as viral vectors, exosomes, molecular Trojanhorses and sundry nanoparticle formulations, have been developed toenhance brain delivery. Viral vectors are effective for gene delivery tobrain, but have limitations such as production cost and safety concerns.Exosomes have been utilized to deliver small molecules, proteins andnucleic acids to the brain due to their non-immunogenic nature; however,there still exist many challenges in the isolation methods, cargoloading procedure, in vivo toxicity and pharmacokinetics. The molecularTrojan horse approach, relying on the receptor-specific monoclonalantibodies or peptides to ferry the genetically fused cargo into thebrain, is promising in delivery of biologics across the BBB. However,the manufacturing process needs to be tailored specifically for each fordifferent biologic cargo, and the stability, safety and immunogenicityare challenge to clinical development. Crossing the BBB with variousnanoparticles, such as liposomes, cationic polymers, inorganicnanoparticles and nanocapsules, have shown promise in delivery ofvarious cargos into the CNS, but complicated modifications are alwaysneeded to ensure the particles produced are BBB-permeable.

Neurotransmitters are endogenous chemicals that enableneurotransmission. Notably, some neurotransmitters have beendemonstrated to cross the BBB. For example, dimethyltryptamine and othertryptamine derivatives have been shown to cross the BBB by activetransport across the endothelial plasma membrane.

SUMMARY

Disclosed herein is a simple, and effective approach for deliveringcargos into brain using neurotransmitter-derived synthetic lipids. Thisapproach is very robust, and can be used to successfully deliverdifferent classes of cargos (small molecule, nucleic acid, and protein,etc.) all using the same, simple nanoparticle design.

In one aspect, disclosed are compounds of formula:

Y—W—R^(Lipid)   (I),

or a pharmaceutically acceptable salt thereof, wherein

-   Y is a moiety derived from a neurotransmitter;-   W is —NR²⁰—, —O—, or —S—;-   R^(Lipid) is independently substituted or unsubstituted C₁₋₂₀ alkyl,    substituted or unsubstituted C₁₋₂₀ alkenyl, substituted or    unsubstituted C₁₋₂₀ alknyl, substituted or unsubstituted C₁₋₂₀    heteroalkyl, substituted or unsubstituted C₁₋₂₀ heteroalkenyl, or    substituted or unsubstituted C₁₋₂₀ heteroalknyl; and-   R²⁰ is R^(Lipid), H, C₁₋₆ alkyl, C₁₋₆ alkenyl, or C₁₋₆ alkynyl.

In certain aspects, disclosed are lipidoid nanoparticles comprising acompound disclosed herein.

In certain aspects, disclosed are pharmaceutical compositions comprisinga lipidoid nanoparticle disclosed herein; and a pharmaceuticallyacceptable carrier or excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic illustration of formulating NT-lipidoid dopedLNPs for cargo delivery to brain.

FIG. 1B is a schematic illustration of synthesis route, lipidnomenclature, and chemical structure of neurotransmitters used forlipidoid synthesis.

FIG. 1C is a representative ex vivo fluorescence images of the dissectedbrain 1 h after one-time intravenous injection of 1 mg kg⁻¹ DiR-labeledNT-LNPs. DiR was doped into the NT-LNPs with a 10% weight ratio. Themice were perfused with saline before dissection.

FIG. 2A is chemical structure of PBA-Q76-O16B, NT1-O12B, and schematicillustration of the doped NT1-lipidoid AmB formulation.

FIG. 2B are photographs of AmB formulations in NT1-O12B doped withdifferent amounts of PBA-Q76O16B (weight ratio is used). The pureNT1-O12B/AmB encapsulates appeared as an opaque suspension, while theappearance of the encapsulates changed from translucent solutions tohomogenous transparent yellow solutions as the doping ratio ofPBA-Q76-O16B lipidoid increased.

FIG. 2C is graph depicting hydrodynamic diameters and polydispersityindexes of different NT-LNP/AmB formulations determined by DLSmeasurements.

FIG. 2D are representative fluorescence images of the dissected mousebrain 1 h after one-time intravenous injection of 1 mg kg⁻¹ DiR-loadedNT1-O12B/PBA-Q76-O16B LNPs. The weight ratio of DiR in LNPs is 10%.

FIG. 2E is a graph depicting AmB concentration in brain tissues 24 hrafter intravenous injection of 5 mg/kg AmB in variousNT1-O12B/PBA-Q76-O16B LNPs LNP formulations measured using HPLC (n=4 pergroup). The mice were perfused with saline before dissected. One-wayANOVA, Sidak post hoc analysis, *p<0.05, **p<0.001 or ***p<0.0001.Graphical data are represented as box and whisker plots with individualpoints overlaid, where error bars represent maximum and minimum valuesand the boxed line represents the median.

FIG. 3A shows chemical structures of 306-O12B-3, NT1-O14B, and schematicillustration of the doped NT-lipidoid Tau-ASO formulation for braindelivery.

FIG. 3B is a graph depicting GFP silencing efficiency of HEK-GFP cellstreated with or without ASO/NT-LNPs complexes. The NT1-O14B LNPs aloneshowed no silencing efficacy, while doping NT1-lipidoid into 306-O12B-3LNPs led to successful gene silencing in vitro. *p<0.01 vs. all othersamples in the same group.

FIG. 3C is a graph depicting Tau-ASOs formulated with NT1-O14B dopedwith different ratios of 306-O12B-3, saline, or scrambled Tau-ASO-LNPswere intravenous injected into C57BL/6J mice (n=6 per group) via thetail vein, and the brain was analyzed for total tau mRNA levels.Graphical data are represented as box and whisker plots with individualpoints overlaid, where error bars represent maximum and minimum valuesand the boxed line represents the median, *p<0.05 or **p<0.001.

FIG. 3D is graph depicting total tau protein levels of theNT1-O14B/306-O12B-3=3:7 group, comparing to that of saline or scrambledTau-ASO, **p<0.001. One-way ANOVA, Sidak post hoc analysis.

FIG. 4A is a schematic illustration of mixed LNP formulation usingNT1-O14B and PBA-Q76-O16B for GFP-Cre protein delivery into brain.

FIG. 4B are fluorescence images of the brain slices of Ai14 mice treatedwith (−27)GFP-Cre in different LNP formulations. Ai14 mouse wasintravenous injected with (−27)GFP-Cre complexed withNT1-O14B/PBA-Q76-O16B=3:7, 10:0 or 0:10 LNPs. After 3 weeks, the groupof NT1-O14B/PBA-Q76-O16B=3:7 showed tdTomato expression indicative ofCre-mediated recombination in cerebral cortex, hippocampus andcerebellum. Scale bar: 100 μm.

FIG. 5 are TEM images of NT1-LNPs and table of hydrodynamic sizes,polydispersity index, zeta potential.

FIG. 6 is graph summarizing relative fluorescence intensity of thedissected brain tissue 1 h after one-time intravenous injection of 1 mgkg−1 DiR-labeled NT-LNPs. DiR was doped into the NT-LNPs with a 10%weight ratio. The mice were perfused with saline before dissection.One-way ANOVA, Sidak post hoc analysis, *p<0.05 or **p<0.01.

FIG. 7 are representative ex vivo fluorescence images of the dissectedbrain 1 h after one-time intravenous injection of 1 mg kg−1 DiR-labeledLNPs or NT1-O12B doped NTLNPs (ratio 3:7, w/w), and the chemicalstructure of 76-O16B, EC16-80, and 113-O16B. DiR was doped into theNT-LNPs with a 10% weight ratio. The mice were perfused with salinebefore dissection.

FIG. 8 depicts the chemical structures of NT-lipidoids anddimethyltryptamine, and the representative ex vivo fluorescence imagesof the dissected brain 1 h after one-time intravenous injection of 1 mgkg−1 DiR-labeled NT-LNPs. DiR was doped into the NTLNPs with a 10%weight ratio. The mice were perfused with saline before dissection.

FIG. 9 is a graph depicting AmB concentration in brain tissues 24 hafter intravenous injection of 5 mg/kg AmB in various NT1 derivativesmeasured using HPLC. The mice were perfused with saline beforedissection.

FIG. 10A is a photograph of AmB formulations in NT1-lipidoids withdifferent tail lengths (O18B, O16B, O14B, O12B). All four NT1/AmBencapsulates showed opaque suspension.

FIG. 10B is a graph depicting hydrodynamic diameters and polydispersityindexes of NT-LNPs determined by DLS measurements.

FIG. 11 is a TEM image of NT1-O12B/PBA-Q76O16B-3/7-AmB complex, and atable that summarizes hydrodynamic sizes, polydispersity index, zetapotential, and DLC of AmB/NT-LNPs complex.

FIG. 12 is a graph that summarizes relative fluorescence intensity ofthe dissected brain tissue 1 h after one-time intravenous injection 1 mgkg−1 DiR-loaded NT1-O12B/PBA-Q76-O16B LNPs. The weight ratio of DiR inLNPs is 10%. **p<0.001. One-way ANOVA, Sidak post hoc analysis.

FIG. 13 is a calibration curve of AmB concentration dissolved inmethanol ranging from 0.005 to 0.5 ug/mL (low concentration), or 0.007to 3.0 ug/mL (high concentration) at 415 nm by HPLC.

FIG. 14 is mAU-time graphs of AmB concentrations in brain tissues 24hafter intravenous treatment with NT1-O12B/PBA-Q76O16-LNPs (ratio:3/7)-AmB complex at a single dose of 5 mg AmB/kg by HPLC.

FIG. 15 are graphs depicting AmB concentrations in other organs 24 hafter intravenous injection of 5 mg/kg AmB measured by HPLC.

FIG. 16 is TEM images of blank and ASO loaded NT1-O14B/306-O12B-3(ratio: 3/7) nanoparticles and a table of hydrodynamic sizes,polydispersity index, zeta potential.

FIG. 17 are TEM images of blank and (−27)GFP-Cre loadedNT1-O14B/PBA-Q76O16B (ratio: 3/7) nanoparticles and a table ofhydrodynamic sizes, polydispersity index, zeta potential.

FIG. 18A is a scheme that depicts the synthesis of 1 E tail.

FIG. 18B is a scheme that depicts the synthesis of PBA-Q76O16B andPBA-Q80O16B.

FIG. 18C is a scheme that depicts the synthesis of NT1-Neu.

FIGS. 19A-19N are fluorescence images of section of Ai14 mouse brain.The mouse were injected with Cre mRNA complexed with Dlin-MC3/NT1-O14BLNP. The LNP formulation was described in slide 1.

FIGS. 20A-20B are fluorescence images of section of Ai14 mouse brain.The mice were injected with Cre mRNA complexed with PBA-Q76O16B/NT1-O14BLNP. The LNP formulation was described in slide 1.

FIGS. 21A-21B are fluorescence images of section of Ai14 mouse brain.The mice were injected with Cre mRNA complexed with Dlin-MC3/NT1-O14BLNP. The LNP formulation was described in slide 1.

DETAILED DESCRIPTION

In one aspect, disclosed are compounds of formula I:

Y—W—R^(Lipid)   (I),

or a pharmaceutically acceptable salt thereof, wherein

-   Y is a moiety derived from a neurotransmitter;-   W is —NR²⁰—, —O—, or —S—;-   R^(Lipid) is independently substituted or unsubstituted C₁₋₂₀ alkyl,    substituted or unsubstituted C₁₋₂₀ alkenyl, substituted or    unsubstituted C₁₋₂₀ alknyl, substituted or unsubstituted C₁₋₂₀    heteroalkyl, substituted or unsubstituted C₁₋₂₀ heteroalkenyl, or    substituted or unsubstituted C₁₋₂₀ heteroalknyl; and-   R²⁰ is R^(Lipid), H, C₁₋₆ alkyl, C₁₋₆ alkenyl, or C₁₋₆ alkynyl.

In certain embodiments, Y is selected from:

In certain preferred embodiments, Y is

In certain embodiments, W is —NR²⁰— or —S—. In certain embodiments, W is—NR²⁰—. In certain embodiments, W is —S—.

In certain embodiments, W is —NR²⁰—, and R²⁰ is R^(Lipid).

In certain embodiments, W is —NR²⁰—, and R²⁰ is R^(Lipid), and Y is

In certain embodiments, R^(Lipid) is of the structure:

-   wherein:-   each instance of R¹ and R² is independently —H, —OH, —NHR³⁰, or —SH;-   R³ and R⁴ are both —H; or R³ and R⁴ are taken together to form an    oxo (═O) group;-   Z is —CH₂—, —O—, —NR³⁰—, or —S—;-   X and Y are independently —CH₂—, —NR³⁰—, —O—, —S—, or —Se—;-   m is an integer selected from 1-3;-   n is an integer selected from 1-14;-   p is 0 or 1;-   q is an integer selected from 1-10;-   t is 0 or 1; and-   R³⁰ is —H, C₁₋₆ alkyl, C₁₋₆ alkenyl, or C₁₋₆ alkynyl.

In certain embodiments, each instance of R¹ and R² is independently —Hor —OH. In certain embodiments, R¹ and R² are —H. In certainembodiments, R¹ is —H; and R² is —OH.

In certain embodiments, R³ and R⁴ are —H. In certain embodiments, R³ andR⁴ are taken together to form an oxo (═O) group.

In certain embodiments, Z is —CH₂—, —O—, or —NR³⁰—. In certainembodiments, Z is —CH₂—. In certain embodiments, Z is —O—. In certainembodiments, Z is —NR³⁰—.

In certain embodiments, R¹ and R² are —H, R³ and R⁴ are taken togetherto form an oxo (═O) group, and Z is O.

In certain embodiments, R¹ is —H, R² is —OH, R³ and R⁴ are —H, and Z is—CH₂—.

In certain embodiments, X and Y are independently —CH₂— or —O—. Incertain embodiments, X and Y are independently —CH₂— or —O—, wherein Xand Y are not the same. In certain embodiments, X and Y areindependently —CH₂— or —S—. In certain embodiments, X and Y are both—CH₂—. In certain embodiments, X and Y are both —S—.

In certain embodiments, m is 1 or 2. In certain embodiments, m is 1. Incertain embodiments, m is 2.

In certain embodiments, n is an integer selected from 4-12. In certainembodiments, n is an integer selected from 6-10.

In certain embodiments, p is 0. In certain embodiments, p is 1.

In certain embodiments, q is an integer selected from 2-8. In certainembodiments, q is an integer selected from 4-8.

In certain embodiments, t is 0. In certain embodiments, t is 1.

In certain embodiments, the compound is selected from the groupconsisting of:

or a pharmaceutically acceptable salt thereof.

In certain aspects, disclosed are lipidoid nanoparticles comprising acompound disclosed herein.

In certain embodiments, the nanoparticle disclosed herein furthercomprising a protein.

In certain embodiments, the protein is GFP-Cre.

In certain embodiments, the nanoparticle disclosed herein furthercomprises a nucleic acid.

In certain embodiments, the nucleic acid is Tau-ASOs.

In certain embodiments, the nanoparticle disclosed herein furthercomprises a small molecule.

In certain embodiments, the small molecule is an antifungal agent or achemotherapeutic agent.

In certain embodiments, the small molecule is selected from the groupconsisting of bortezomib, imatinib, gefitinib, erlotinib, afatinib,osimertinib, dacomitinib, daunorubicin hydrochloride, cytarabine,fluorouracil, irinotecan hydrochloride, vincristine sulfate,methotrexate, paclitaxel, vincristine sulfate, epirubicin, docetaxel,cyclophosphamide, carboplatin, lenalidomide, ibrutinib, abirateroneacetate, enzalutamide, pemetrexed, palbociclib, nilotinib, everolimus,ruxolitinib, epirubicin, pirirubicin, idarubicin, valrubicin, amrubicin,bleomycin, phleomycin, dactinomycin, mithramycin, streptozotecin,pentostatin, mitosanes mitomycin C, enediynes calicheamycin, glycosidesrebeccamycin, macrolide lactones epotihilones, ixabepilone, pentostatin,salinosporamide A, vinblastine, vincristine, etoposide, teniposide,vinorelbine, docetaxel, camptothecin, hycamtin, pederin, theopederins,annamides, trabectedin, aplidine, and ecteinascidin 743 (ET743).

In certain embodiments, the small molecule is amphotericin B ordoxorubicin.

In certain embodiments, the lipidoid nanoparticle has a particle size ofabout 25 nm to about 1000 nm. In certain embodiments, the lipidoidnanoparticle has a particle size of about 50 nm to about 500 nm.

In certain aspects, disclosed are pharmaceutical compositions comprisinga lipidoid nanoparticle disclosed herein; and a pharmaceuticallyacceptable carrier or excipient.

Definitions

Unless otherwise defined herein, scientific and technical terms used inthis application shall have the meanings that are commonly understood bythose of ordinary skill in the art. Generally, nomenclature used inconnection with, and techniques of, chemistry, cell and tissue culture,molecular biology, cell and cancer biology, neurobiology,neurochemistry, virology, immunology, microbiology, pharmacology,genetics and protein and nucleic acid chemistry, described herein, arethose well-known and commonly used in the art.

The methods and techniques of the present disclosure are generallyperformed, unless otherwise indicated, according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout thisspecification. See, e.g. “Principles of Neural Science”, McGraw-HillMedical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”,Oxford University Press, Inc. (1995); Lodish et al., “Molecular CellBiology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths etal., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co.,N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”,Sinauer Associates, Inc., Sunderland, Mass. (2000).

Chemistry terms used herein, unless otherwise defined herein, are usedaccording to conventional usage in the art, as exemplified by “TheMcGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill,San Francisco, Calif. (1985).

As used herein, the terms “optional” or “optionally” mean that thesubsequently described event or circumstance may occur or may not occur,and that the description includes instances where the event orcircumstance occurs as well as instances in which it does not. Forexample, “optionally substituted alkyl” refers to the alkyl may besubstituted as well as where the alkyl is not substituted.

It is understood that substituents and substitution patterns on thecompounds of the present invention can be selected by one of ordinaryskilled person in the art to result chemically stable compounds whichcan be readily synthesized by techniques known in the art, as well asthose methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.

As used herein, the term “optionally substituted” refers to thereplacement of one to six hydrogen radicals in a given structure withthe radical of a specified substituent including, but not limited to:hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl,acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano,haloalkyl, haloalkoxy, —OCO—CH₂—O-alkyl, —OP(O)(O-alkyl)₂ or—CH₂—OP(O)(O-alkyl)₂. Preferably, “optionally substituted” refers to thereplacement of one to four hydrogen radicals in a given structure withthe substituents mentioned above. More preferably, one to three hydrogenradicals are replaced by the substituents as mentioned above. It isunderstood that the substituent can be further substituted.

Articles such as “a,” “an,” and “the” may mean one or more than oneunless indicated to the contrary or otherwise evident from the context.Claims or descriptions that include “or” between one or more members ofa group are considered satisfied if one, more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process unless indicated to the contrary or otherwiseevident from the context. The invention includes embodiments in whichexactly one member of the group is present in, employed in, or otherwiserelevant to a given product or process. The invention includesembodiments in which more than one, or all of the group members arepresent in, employed in, or otherwise relevant to a given product orprocess.

As used herein, the term “alkyl” refers to saturated aliphatic groups,including but not limited to C₁-C₁₀ straight-chain alkyl groups orC₁-C₁₀ branched-chain alkyl groups. Preferably, the “alkyl” group refersto C₁-C₆ straight-chain alkyl groups or C₁-C₆ branched-chain alkylgroups. Most preferably, the “alkyl” group refers to C₁-C₄straight-chain alkyl groups or C₁-C₄ branched-chain alkyl groups.Examples of “alkyl” include, but are not limited to, methyl, ethyl,1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl,3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl,3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl or 4-octyl and the like.The “alkyl” group may be optionally substituted.

The term “acyl” is art-recognized and refers to a group represented bythe general formula hydrocarbylC(O)—, preferably alkylC(O)—.

The term “acylamino” is art-recognized and refers to an amino groupsubstituted with an acyl group and may be represented, for example, bythe formula hydrocarbylC(O)NH—.

The term “acyloxy” is art-recognized and refers to a group representedby the general formula hydrocarbylC(O)O—, preferably alkylC(O)O—.

The term “alkoxy” refers to an alkyl group having an oxygen attachedthereto. Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group and may be represented by the general formulaalkyl-O-alkyl.

The term “alkyl” refers to saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁₋₃₀ for straight chains, C₃₋₃₀ for branchedchains), and more preferably 20 or fewer.

Moreover, the term “alkyl” as used throughout the specification,examples, and claims is intended to include both unsubstituted andsubstituted alkyl groups, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone, including haloalkyl groups such as trifluoromethyland 2,2,2-trifluoroethyl, etc.

The term “C_(x-y)” or “C_(x)-C_(y)”, when used in conjunction with achemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, oralkoxy is meant to include groups that contain from x to y carbons inthe chain. C₀alkyl indicates a hydrogen where the group is in a terminalposition, a bond if internal. A C₁₋₆alkyl group, for example, containsfrom one to six carbon atoms in the chain.

The term “alkylamino”, as used herein, refers to an amino groupsubstituted with at least one alkyl group.

The term “alkylthio”, as used herein, refers to a thiol groupsubstituted with an alkyl group and may be represented by the generalformula alkylS—.

The term “amide”, as used herein, refers to a group

wherein R⁹ and R¹⁰ each independently represent a hydrogen orhydrocarbyl group, or R⁹ and R¹⁰ taken together with the N atom to whichthey are attached complete a heterocycle having from 4 to 8 atoms in thering structure.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

wherein R⁹, R¹⁰, and R¹⁰, each independently represent a hydrogen or ahydrocarbyl group, or R⁹ and R¹⁰ taken together with the N atom to whichthey are attached complete a heterocycle having from 4 to 8 atoms in thering structure.

The term “aminoalkyl”, as used herein, refers to an alkyl groupsubstituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group.

The term “aryl” as used herein include substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably, the ring is a 5- to 7-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike.

The term “carbamate” is art-recognized and refers to a group

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbylgroup.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a carbocycle group.

The term “carbocycle” includes 5-7 membered monocyclic and 8-12 memberedbicyclic rings. Each ring of a bicyclic carbocycle may be selected fromsaturated, unsaturated and aromatic rings. Carbocycle includes bicyclicmolecules in which one, two or three or more atoms are shared betweenthe two rings. The term “fused carbocycle” refers to a bicycliccarbocycle in which each of the rings shares two adjacent atoms with theother ring. Each ring of a fused carbocycle may be selected fromsaturated, unsaturated and aromatic rings. In an exemplary embodiment,an aromatic ring, e.g., phenyl, may be fused to a saturated orunsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Anycombination of saturated, unsaturated and aromatic bicyclic rings, asvalence permits, is included in the definition of carbocyclic. Exemplary“carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane,1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene,bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary fusedcarbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene,bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene andbicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted at any one ormore positions capable of bearing a hydrogen atom.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a carbocycle group.

The term “carbonate” is art-recognized and refers to a group —OCO₂—.

The term “carboxy”, as used herein, refers to a group represented by theformula —CO₂H.

The term “ester”, as used herein, refers to a group —C(O)OR⁹ wherein R⁹represents a hydrocarbyl group.

The term “ether”, as used herein, refers to a hydrocarbyl group linkedthrough an oxygen to another hydrocarbyl group. Accordingly, an ethersubstituent of a hydrocarbyl group may be hydrocarbyl-O—. Ethers may beeither symmetrical or unsymmetrical. Examples of ethers include, but arenot limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethersinclude “alkoxyalkyl” groups, which may be represented by the generalformula alkyl-O-alkyl.

The terms “halo” and “halogen” as used herein means halogen and includeschloro, fluoro, bromo, and iodo.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to analkyl group substituted with a hetaryl group.

The terms “heteroaryl” and “hetaryl” include substituted orunsubstituted aromatic single ring structures, preferably 5- to7-membered rings, more preferably 5- to 6-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heteroaryl” and “hetaryl” also include polycyclic ring systems havingtwo or more cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine,and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a heterocycle group.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer tosubstituted or unsubstituted non-aromatic ring structures, preferably 3-to 10-membered rings, more preferably 3- to 7-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heterocyclyl” and “heterocyclic” also include polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings isheterocyclic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Heterocyclyl groups include, for example, piperidine, piperazine,pyrrolidine, morpholine, lactones, lactams, and the like.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom that does not have a ═O or ═S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but may optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are consideredto be hydrocarbyl for the purposes of this application, but substituentssuch as acetyl (which has a ═O substituent on the linking carbon) andethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbylgroups include, but are not limited to aryl, heteroaryl, carbocycle,heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “hydroxyalkyl”, as used herein, refers to an alkyl groupsubstituted with a hydroxy group.

The term “lower” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups where there are ten or fewer atoms in the substituent,preferably six or fewer. A “lower alkyl”, for example, refers to analkyl group that contains ten or fewer carbon atoms, preferably six orfewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl,or alkoxy substituents defined herein are respectively lower acyl, loweracyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy,whether they appear alone or in combination with other substituents,such as in the recitations hydroxyalkyl and aralkyl (in which case, forexample, the atoms within the aryl group are not counted when countingthe carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more atoms are commonto two adjoining rings, e.g., the rings are “fused rings”. Each of therings of the polycycle can be substituted or unsubstituted. In certainembodiments, each ring of the polycycle contains from 3 to 10 atoms inthe ring, preferably from 5 to 7.

The term “sulfate” is art-recognized and refers to the group —OSO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfonamide” is art-recognized and refers to the grouprepresented by the general formulae

wherein R⁹ and R¹⁰ independently represents hydrogen or hydrocarbyl.

The term “sulfoxide” is art-recognized and refers to the group—S(O)—.

The term “sulfonate” is art-recognized and refers to the group SO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfone” is art-recognized and refers to the group —S(O)₂—.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate.

The term “thioalkyl”, as used herein, refers to an alkyl groupsubstituted with a thiol group.

The term “thioester”, as used herein, refers to a group —C(O)SR⁹ or—SC(O)R⁹

wherein R⁹ represents a hydrocarbyl.

The term “thioether”, as used herein, is equivalent to an ether, whereinthe oxygen is replaced with a sulfur.

The term “urea” is art-recognized and may be represented by the generalformula

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbyl.

The term “modulate” as used herein includes the inhibition orsuppression of a function or activity (such as cell proliferation) aswell as the enhancement of a function or activity.

The phrase “pharmaceutically acceptable” is art-recognized. In certainembodiments, the term includes compositions, excipients, adjuvants,polymers and other materials and/or dosage forms which are, within thescope of sound medical judgment, suitable for use in contact with thetissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

“Salt” is used herein to refer to an acid addition salt or a basicaddition salt.

Many of the compounds useful in the methods and compositions of thisdisclosure have at least one stereogenic center in their structure. Thisstereogenic center may be present in a R or a S configuration, said Rand S notation is used in correspondence with the rules described inPure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates allstereoisomeric forms such as enantiomeric and diastereoisomeric forms ofthe compounds, salts, prodrugs or mixtures thereof (including allpossible mixtures of stereoisomers). See, e.g., WO 01/062726.

Furthermore, certain compounds which contain alkenyl groups may exist asZ (zusammen) or E (entgegen) isomers. In each instance, the disclosureincludes both mixture and separate individual isomers.

Some of the compounds may also exist in tautomeric forms. Such forms,although not explicitly indicated in the formulae described herein, areintended to be included within the scope of the present disclosure.

“Pharmaceutically acceptable” means approved or approvable by aregulatory agency of the Federal or a state government or thecorresponding agency in countries other than the United States, or thatis listed in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and more particularly, in humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound of theinvention that is pharmaceutically acceptable and that possesses thedesired pharmacological activity of the parent compound. In particular,such salts are non-toxic may be inorganic or organic acid addition saltsand base addition salts. Specifically, such salts include: (1) acidaddition salts, formed with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike; or formed with organic acids such as acetic acid, propionic acid,hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-l-carboxylic acid, glucoheptonic acid ,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid , gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion , an alkalineearth ion , or an aluminum ion; or coordinates with an organic base suchas ethanolamine, diethanolamine, triethanolamine, N-methylglucamine andthe like. Salts further include, by way of example only, sodiumpotassium, calcium, magnesium, ammonium, tetraalkylammonium, and thelike; and when the compound contains a basic functionality, salts ofnontoxic organic or inorganic acids, such as hydrochloride,hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and thelike.

The term “pharmaceutically acceptable cation” refers to an acceptablecationic counterion of an acidic functional group. Such cations areexemplified by sodium, potassium, calcium, magnesium, ammonium,tetraalkylammonium cations, and the like (see, e. g., Berge, et al., J.Pharm. Sci. 66 (1):1-79 (January 77).

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient or carrier with which a compound of the invention isadministered.

“Pharmaceutically acceptable metabolically cleavable group” refers to agroup that is cleaved in vivo to yield the parent molecule of thestructural formula indicated herein. Examples of metabolically cleavablegroups include —COR, —COOR, —CONRR and —CH₂OR radicals, where R isselected independently at each occurrence from alkyl, trialkylsilyl,carbocyclic aryl or carbocyclic aryl substituted with one or more ofalkyl, halogen, hydroxy or alkoxy. Specific examples of representativemetabolically cleavable groups include acetyl, methoxycarbonyl, benzoyl,methoxymethyl and trimethylsilyl groups.

“Prodrugs” refers to compounds, including derivatives of the compoundsof the invention, which have cleavable groups and become by solvolysisor under physiological conditions the compounds of the invention whichare pharmaceutically active in vivo. Such examples include, but are notlimited to, choline ester derivatives and the like, N-alkylmorpholineesters and the like. Other derivatives of the compounds of thisinvention have activity in both their acid and acid derivative forms,but in the acid sensitive form often offers advantages of solubility,tissue compatibility, or delayed release in the mammalian organism (see,Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam1985). Prodrugs include acid derivatives well known to practitioners ofthe art, such as, for example, esters prepared by reaction of the parentacid with a suitable alcohol, or amides prepared by reaction of theparent acid compound with a substituted or unsubstituted amine, or acidanhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters,amides and anhydrides derived from acidic groups pendant on thecompounds of this invention are particular prodrugs. In some cases it isdesirable to prepare double ester type prodrugs such as(acyloxy)alkylesters or (alkoxycarbonyl)oxy)alkylesters. Particularlythe C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, aryl, C₇-C₁₂ substitutedaryl, and C₇-C₁₂ arylalkyl esters of the compounds of the invention.

“Solvate” refers to forms of the compound that are associated with asolvent or water (also referred to as “hydrate”), usually by asolvolysis reaction. This physical association includes hydrogenbonding. Conventional solvents include water, ethanol, acetic acid andthe like. The compounds of the invention may be prepared e.g., incrystalline form and may be solvated or hydrated. Suitable solvatesinclude pharmaceutically acceptable solvates, such as hydrates, andfurther include both stoichiometric solvates and non-stoichiometricsolvates. In certain instances, the solvate will be capable ofisolation, for example when one or more solvent molecules areincorporated in the crystal lattice of the crystalline solid. “Solvate”encompasses both solution-phase and isolable solvates. Representativesolvates include hydrates, ethanolates and methanolates.

A “subject” to which administration is contemplated includes, but is notlimited to, humans (i.e., a male or female of any age group, e.g., apediatric subject (e.g, infant, child, adolescent) or adult subject(e.g., young adult, middle aged adult or senior adult) and/or anon-human animal, e.g., a mammal such as primates (e.g., cynomolgusmonkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents,cats, and/or dogs. In certain embodiments, the subject is a human. Incertain embodiments, the subject is a non-human animal. The terms“human,” “patient,” and “subject” are used interchangeably herein.

An “effective amount” means the amount of a compound that, whenadministered to a subject for treating or preventing a disease, issufficient to effect such treatment or prevention. The “effectiveamount” can vary depending on the compound, the disease and itsseverity, and the age, weight, etc., of the subject to be treated. A“therapeutically effective amount” refers to the effective amount fortherapeutic treatment. A “prophylatically effective amount” refers tothe effective amount for prophylactic treatment.

“Preventing” or “prevention” or “prophylactic treatment” refers to areduction in risk of acquiring or developing a disease or disorder(i.e., causing at least one of the clinical symptoms of the disease notto develop in a subject not yet exposed to a disease-causing agent, orpredisposed to the disease in advance of disease onset.

The term “prophylaxis” is related to “prevention,” and refers to ameasure or procedure the purpose of which is to prevent, rather than totreat or cure a disease. Non limiting examples of prophylactic measuresmay include the administration of vaccines; the administration of lowmolecular weight heparin to hospital patients at risk for thrombosisdue, for example, to immobilization, and the administration of ananti-malarial agent such as chloroquine, in advance of a visit to ageographical region where malaria is endemic or the risk of contractingmalaria is high.

“Treating” or “treatment” or “therapeutic treatment” of any disease ordisorder refers, in one embodiment, to ameliorating the disease ordisorder (i.e., arresting the disease or reducing the manifestation,extent or severity of at least one of the clinical symptoms thereof). Inanother embodiment “treating” or “treatment” refers to ameliorating atleast one physical parameter, which may not be discernible by thesubject. In yet another embodiment, “treating” or “treatment” refers tomodulating the disease or disorder, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both. In a furtherembodiment, “treating” or “treatment” relates to slowing the progressionof the disease.

As used herein, the term “isotopic variant” refers to a compound thatcontains unnatural proportions of isotopes at one or more of the atomsthat constitute such compound. For example, an “isotopic variant” of acompound can contain one or more non-radioactive isotopes, such as forexample, deuterium (²H or D), carbon-13 (¹³C), nitrogen-15 (¹⁵N), or thelike. It will be understood that, in a compound where such isotopicsubstitution is made, the following atoms, where present, may vary, sothat for example, any hydrogen may be “²H/D, any carbon may be ¹³C, orany nitrogen may be ¹⁵N, and that the presence and placement of suchatoms may be determined within the skill of the art. Likewise, theinvention may include the preparation of isotopic variants withradioisotopes, in the instance for example, where the resultingcompounds may be used for drug and/or substrate tissue distributionstudies. The radioactive isotopes tritium, i.e., ³H, and carbon-14,i.e., ¹⁴C, are particularly useful for this purpose in view of theirease of incorporation and ready means of detection. Further, com poundsmay be prepared that are substituted with positron emitting isotopes,such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, and would be useful in Positron EmissionTopography (PET) studies for examining substrate receptor occupancy. Allisotopic variants of the compounds provided herein, radioactive or not,are intended to be encompassed within the scope of the invention.

It is also to be understood that compounds that have the same molecularformula but differ in the nature or sequence of bonding of their atomsor the arrangement of their atoms in space are termed “isomers.” Isomersthat differ in the arrangement of their atoms in space are termed“stereoisomers.”

Stereoisomers that are not mirror images of one another are termed“diastereomers” and those that are non-superimposable mirror images ofeach other are termed “enantiomers.” When a compound has an asymmetriccenter, for example, it is bonded to four different groups, a pair ofenantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is described by theR—and S—sequencing rules of Cahn and Prelog, or by the manner in whichthe molecule rotates the plane of polarized light and designated asdextrorotatory or levorotatory (i.e., as (+)- or (−)-isomersrespectively). A chiral compound can exist as either individualenantiomer or as a mixture thereof. A mixture containing equalproportions of the enantiomers is called a “racemic mixture”.

“Tautomers” refer to compounds that are interchangeable forms of aparticular compound structure, and that vary in the displacement ofhydrogen atoms and electrons. Thus, two structures may be in equilibriumthrough the movement of it electrons and an atom (usually H). Forexample, enols and ketones are tautomers because they are rapidlyinterconverted by treatment with either acid or base. Another example oftautomerism is the aci- and nitro-forms of phenylnitromethane, that arelikewise formed by treatment with acid or base. Tautomeric forms may berelevant to the attainment of the optimal chemical reactivity andbiological activity of a compound of interest.

As used herein a pure enantiomeric compound is substantially free fromother enantiomers or stereoisomers of the compound (i.e., inenantiomeric excess). In other words, an “S” form of the compound issubstantially free from the “R” form of the compound and is, thus, inenantiomeric excess of the “R” form. The term “enantiomerically pure” or“pure enantiomer” denotes that the compound comprises more than 95% byweight, more than 96% by weight, more than 97% by weight, more than 98%by weight, more than 98.5% by weight, more than 99% by weight, more than99.2% by weight, more than 99.5% by weight, more than 99.6% by weight,more than 99.7% by weight, more than 99.8% by weight or more than 99.9%by weight, of the enantiomer. In certain embodiments, the weights arebased upon total weight of all enantiomers or stereoisomers of thecompound.

As used herein and unless otherwise indicated, the term“enantiomerically pure R-compound” refers to at least about 95% byweight R-compound and at most about 5% by weight S-compound, at leastabout 99% by weight R-compound and at most about 1% by weightS-compound, or at least about 99.9% by weight R-compound and at mostabout 0.1% by weight S-compound. In certain embodiments, the weights arebased upon total weight of compound.

As used herein and unless otherwise indicated, the term“enantiomerically pure 5-compound” or “S-compound” refers to at leastabout 95% by weight S-compound and at most about 5% by weightR-compound, at least about 99% by weight S-compound and at most about 1%by weight R-compound or at least about 99.9% by weight S-compound and atmost about 0.1% by weight R-compound. In certain embodiments, theweights are based upon total weight of compound.

In the compositions provided herein, an enantiomerically pure compoundor a pharmaceutically acceptable salt, solvate, hydrate or prodrugthereof can be present with other active or inactive ingredients. Forexample, a pharmaceutical composition comprising enantiomerically pureR-compound can comprise, for example, about 90% excipient and about 10%enantiomerically pure R-compound. In certain embodiments, theenantiomerically pure R-compound in such compositions can, for example,comprise at least about 95% by weight R-compound and at most about 5% byweight S-compound, by total weight of the compound. For example, apharmaceutical composition comprising enantiomerically pure S-compoundcan comprise, for example, about 90% excipient and about 10%enantiomerically pure S-compound. In certain embodiments, theenantiomerically pure S-compound in such compositions can, for example,comprise, at least about 95% by weight S-compound and at most about 5%by weight R-compound, by total weight of the compound. In certainembodiments, the active ingredient can be formulated with little or noexcipient or carrier.

The compounds of this invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof.

Unless indicated otherwise, the description or naming of a particularcompound in the specification and claims is intended to include bothindividual enantiomers and mixtures, racemic or otherwise, thereof. Themethods for the determination of stereochemistry and the separation ofstereoisomers are well-known in the art.

One having ordinary skill in the art of organic synthesis will recognizethat the maximum number of heteroatoms in a stable, chemically feasibleheterocyclic ring, whether it is aromatic or non-aromatic, is determinedby the size of the ring, the degree of unsaturation and the valence ofthe heteroatoms. In general, a heterocyclic ring may have one to fourheteroatoms so long as the heteroaromatic ring is chemically feasibleand stable.

EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. The examples describedin this application are offered to illustrate the compounds,compositions, materials, device, and methods provided herein and are notto be construed in any way as limiting their scope.

Materials and Methods General:

All chemicals used for lipid synthesis were purchased from Sigma-Aldrichand directly used as received. All ASOs and DNA fragments were purchasedfrom Integrated DNA Technologies (IDT). The ASOs were used as providedfrom IDT, and when noted we used the ASO product that is provided by thecompany to contain chemical modifications to improve stability.HeLa-DsRed and GFP-HEK cells were maintained in Dulbecco's modifiedeagle's medium (DMEM, Sigma-Aldrich) complemented with 10% fetal bovineserum (FBS, Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco). Thefluorescent intensity for GFP-HEK cells was analyzed by flow cytometer(BD FACS Calibur, BD Science, CA). The (−27)GFP-Cre (addgene #89253)protein were expressed and extracted from BL21 E.coli, and furtherpurified by Ni-NTA column (Qiagen). Nanoparticle size and potential wererecorded on ZetaPALS particle size analyzer. TEM images were captured bya FEI Technai Spirit Transmission Electron Microscope.

Lipid Synthesis

All head amines used for lipid synthesis are commercially available fromSigma-Aldrich. All the cationic lipidoids (NT1-O12B˜O18B, NT2-O12B˜O18B,NT3-O12B˜O18B, NT1-EC16, NT1-C18, NT1-1E, NT2-EC16, NT2-1E, NT3-EC16,NT3-1E, 306-O12B-3, 76-O16B) were synthesized according to our previousreports. The crude product was purified using flash chromatography onsilica gel. The 1E tail was synthesized as shown in FIG. 18A. Thephenylboronic acid quaternized lipidoids were synthesized as shown inFIG. 18B. NT1-Neu was synthesized as shown in FIG. 18C. The lipidstructure was confirmed using 1H NMR and electrospray ionization (ESI)MS.

Biodistribution of DiR Labelled NT-LNPs in Mice Brain

The NT-lipidoids and DiR is dissloved at a weight ratio of 10:1,together in 100% ethanol. 100 μL solution was then added dropwise to 300μL of sodium acetate buffer (25 mM, pH 5.2) and vortexed briefly.Finally, we removed the ethanol in the formulation by dialysis againstdiH2O (MWCO 35 kDa, ThermoFisher) for 12 h. Then the DiR-labelled LNPswere intravenously injected into BALB/C mice (female, 6 weeks age).After 1 hour, mice were anesthetized and perfused with saline.Afterward, mice brains were collected. The fluorescent signalsdistribution was visualized using the Spectrum CT Biophotonic Imager(PerkinElmer, Boston, Mass.).

Preparation of AmB/NT-Lipidoid Nanoparticles Formulations

The AmB encapsulates were prepared according to our previous report.3Briefly, 1 mg each lipidoid (solid) was mixed with 1 mg AmB in 300 μLDimethyl sulfoxide (DMSO). The mixtures were sonicated for 30 min andthen vortexed for 10 min until completely dissolved. The solution wasadded drop wise to a glass bottle containing 600 μL sodium acetatebuffer (pH 5.0) with continuous homogenization at 700 rpm. The solutionswere further dialyzed against distilled water to remove DMSO andnon-encapsulated AmB using dialysis tubing (MWCO 35 kDa) overnight.

Characterization of AmB/NT-Lipidoid Nanoparticles Formulations

The particle sizes and polydispersity index (PDI) of all encapsulateswere measured using dynamic light scattering (DLS). potential wererecorded on ZetaPALS particle size analyzer. The DLCs of AmB werecalculated according to to our previous report.3 TEM images werecaptured by a FEI Technai Spirit Transmission Electron Microscope.

Statistical Analysis

Statistical analysis was performed using one-way analysis of variance(ANOVA) followed by the Turkey-Kramer multiple comparison test for morethan two groups. Student t-test were used for comparing two groups usingPrism (v.8, GraphPad Software, La Jolla, Calif.). Values of p<0.05 wereconsidered as significance.

NT-Lipidoids Synthesis and BBB-Permeability Study of the NT-LNPs

Neurotransmitters tryptamine, phenethylamine, and phenylethanolaminewere selected as the structural basis of the synthetic lipidoids. TheNT-lipidoids were synthesized through Michael addition between theprimary amine of the neurotransmitters and acrylate-containinghydrophobic tails in glass vials at 70° C. for 48 h (FIG. 1B), using anapproach similar to our previously published combinatorial lipid librarysynthesis strategy. The result is a combinatorial library ofNT-lipidoids, each containing one particular neurotransmitter as thehead group, and one particular bioreducible hydrophobic structure as thetail group. The NT-lipidoids were named “NTn-O[x]B” (n=1, 2, 3), whereNT1 is tryptamine, NT2 is phenethylamine, NT3 is phenylethanolamine, andO[x]B represents the bioreducible hydrophobic tail where [x] indicatesthe number of carbon atoms in the hydrophobic tail of the acrylate shownin FIG. 1B. For example, NT1-O12B indicates a lipidoid containing atryptamine head group, and a hydrophobic tail group containing 12 carbonatoms. All the NT-lipidoids were purified using flash chromatography andcharacterized by ESI-MS (FIG. 5). The resulting NT-lipidoids areamphiphilic, and thus are capable of self-assembly into either micellesor liposomes when prepared in aqueous solution. Dynamic Light Scattering(DLS) and Transmission Electron Microscopy (TEM) of the NT-lipidoidsindicated that these structures indeed self-assembled into sphericalliposome-like structures (FIG. 6).

TABLE 1 MS Values of the NT-Derived Lipids Synthesized ESI-MS LipidStructure Designation [M + H]+

NT1-O12B 713.61

NT1-O14B 769.61

NT2-O12B 674.61

NT2-O14B 730.62

NT1-O16B 825.70

NT1-O18B 881.75

NT2-O16B 786.64

NT2-O18B 842.72

NT3-O12B 690.55

NT3-O14B 746.65

NT1-Neu 681.52

NT1-1E 925.82

NT1-EC16 641.82

NT1-C18 665.91

NT3-O16B 802.73

NT3-O18B 858.73

NT2-1E 886.82

NT2-EC16 602.82

306-O12B3 974.73

PBA-Q76- Q16B 913.73

NT3-1E 902.91

NT3-EC16 618.82

Whether these NT-lipidoids can cross the BBB upon systemic intravenousdelivery, using a fluorescent dye (DiR) as a model cargo was furtherstudied. Hydrophobic small molecules such as DiR can partition into thehydrophobic region of micelles and liposomes and have often been used totrack the biodistribution of these structures. To formulate theDiR-loaded NT-lipidoids, the NT-lipidoid and DiR were mixed in ethanolin a 10/1 (w/w) ratio, added the mixture dropwise to sodium acetatebuffer (25 mM, pH 5.2), and then removed the ethanol through dialysis.The DiR-loaded NT-lipidoid nanoparticle solution was injected into mousevia tail vein injection. After 1 h, the animals were sacrificed andperfused with saline. The skull was removed, and the brain was imagedusing an IVIS imaging device (PerkinElmer) at the excitation wavelengthof 750 nm.

As shown in FIG. 1C, strong DiR fluorescence signal is observed in themouse brain treated with DiR/NT1-lipidoid nanoparticles, compared to themouse brain treated with DiR/NT2-lipidoids and DiR/NT3-lipidoids wherethe fluorescent signal was very weak. It is also observed that length ofthe aliphatic tail chain significantly influenced the observedfluorescent intensity, with NT1-lipidoids containing a shorter aliphaticchain length resulting in a greater fluorescence intensity (FIG. 7).There is no significant difference of the physical properties, such ashydrodynamic sizes, polydispersity index, zeta potential, andmorphologies between these NT1 derived lipidoids (FIG. 6).

It is hypothesized that doping NT1-lipidoids such as NT1-O12B into otherBBB-impermeable lipid formulations led to the resulting lipidformulation crossing the BBB. The previously published synthetic lipids,76-O16B, EC16-80, and 113-O16B, were used to test this ability todeliver DiR to the brain. It is found that none of these lipids wereeffective in delivering the DiR into the mouse brain by themselves,however, after doping these lipids with the NT1-O12B, strong DiR signalscould be observed in the mouse brain (FIG. 8).

The chemical structure of NT1 is based on the neurotransmitterdimethyltryptamine which has been reported to cross the BBB by activetransport across the endothelial plasma membrane.²¹ It is hypothesizedthat our results are also driven by active transport, and that changesin the chemical structure of the NT1-lipid would modulate its ability tocross the BBB. We hypothesized specifically that the ionizability of thea-amine in the tryptamine (NT1) after the lipidization is an importantfactor for the derivatives to cross BBB. To verify this hypothesis, aseries of NT1 derivatives with different linkers were synthesized, asshown in FIG. 9. The DiR signals were observed from the mouse braintreated with all NT1 derivatives except NT1-neu. In NT1-neu, the a-aminein the tryptamine is connected through an amide bond, not ionizable,while the a-amine in other NT1 derivatives are all ionizable. Further,no strong DiR signals were observed from the mouse brain treated withNT2 and NT3 derived lipidoids with any linkers.

Delivery of Small Molecule AmB into the Mouse Brain

As shown above, NT1 derived lipidoids are identified to be able todeliver a hydrophobic dye (DiR) into brain, either when used alone orwhen doped into other LNPs. Delivering therapeutically relevanthydrophobic drug molecules into brain was examined using theseNT1-derived lipidoids. Amphotericin B (AmB) was chosen as a model drug.AmB is a classic polyene antifungal drug and is the gold standard forthe treatment of severe systemic fungal infections. However, it cannotbe used clinically for the treatment of brain fungal infections due toits BBB-impermeability. Recently, AmB was formulated in syntheticlipidoid nanoparticles and conducted a thorough PK and biodistributionstudy of the AmB formulations using traditional synthetic lipidnanoparticles, but in that study none of our lipid nanoparticles werecapable of permeating the BBB to deliver AmB into the mouse brain.²⁷

AmB was encapsulated in pure NT1-lipidoids (namely NT1-O12B, NT1-O14B,NT1-O16B, and NT1-O18B) using a procedure similar to the DiRencapsulation. The AmB loaded NT1-lipidoid nanoparticles were injectedinto mice via tail vein at a dose of 5 mg/kg AmB per mouse. After 24 hr,animals were sacrificed, and the brains were harvested, perfused withsaline, and homogenized. The AmB concentration in brain tissue wasquantified using HPLC (detailed methods are in SI). As shown in FIG. 10,the AmB concentration in the brain tissue of all the four groups wasaround 150 ng/g tissue. Notably, in our previous report, AmB wasundetectable in the brain after systemic delivery with traditionalsynthetic lipidoids, indicating the NT1-lipidoid formulations enhancedthe AmB delivery to the mouse brain.

However, the AmB formulated in NT-lipidoid formulation showed opaquesolution (FIG. 11A), indicating the large size of the particles in thesolution. DLS results (FIG. 11B, FIG. 12) showed that the nanoparticlesare in range of 750-800 nm in diameter. It is hypothesized that makingNT1-lipidoid nanoparticle smaller may help improve the brain deliveryefficiency. In the previous report, it is found that the quaternizedlipidoids provided stable AmB formulation with smaller particle size,comparing with the non-quaternized lipids.²⁷ Thus, it is hypothesizedthat doping a quaternized lipidoid with NT1-lipidoid may result in asmaller nanoparticle size while maintaining or improving the ability topenetrate the BBB.

Here a new phenylboronic acid quaternized lipidoid was synthesized,PBA-Q76-O16B (FIG. 2A) for AmB encapsulation. NT1-O12B was chosen as thedopant for enhanced brain delivery since it showed the highest DiRfluorescence intensity (FIG. 1C) among all NT lipidoids. The AmB wasformulated in the mixture of NT1-O12B and PBA-Q76-O16B, with the twolipidoids mixed at different weight ratios (7:3, 5:5, 3:7, 1:9, and purePBA-Q76-O16B). As shown in FIG. 2B, the AmB encapsulates graduallybecame homogenous transparent yellow solution with the increasingpercentage of PBA-Q76-O16B lipidoid in the formulations. Thehydrodynamic sizes also decreased from 800 nm to 100 nm (FIG. 2C, FIG.12). Using DiR as a cargo, we observed that lipidoids containingNT1-O12B and PBA-Q76-O16B at a 3:7 (w/w) ratio provided the strongestfluorescent signal in mouse brain when compared with all other lipidratios (FIG. 2D). The fluorescent signal intensity at a 3:7 ratio was4.5 folds higher than that of brain treated with DiR formulated in pureNT1-O12B (FIG. 13). The AmB delivery using the mixed lipids was furtherstudied and determined the AmB concentration in the mice brain tissue 24hr after intravenous injection of 5 mg/kg AmB per mouse. As shown inFIG. 2E, the amount of AmB detected in the brain increased as the dopingratio of PBA-Q76-O16B increased from 0% (i.e. pure NT1-O12B) to 70%(i.e. 3:7 ratio) and reached a highest concentration around 300 ng/g,which was about 2 folds higher than pure NT1-O12B. When the doping ratioincreased further to 90% (i.e. 1:9), the AmB concentration was slightlylower, but was still higher than of that treated with AmB formulated inpure NT1-O12B. Thus, the results for AmB delivery closely matched theresults for DiR delivery (FIGS. 2D and 2E). Interestingly, withoutdoping with NT1-lipidoid, the AmB was nearly undetectable in the brainafter intravenous injection of pure PBA-Q76-O16B/AmB. These resultsshowed the key role of NT1 lipidoids in facilitating the brain delivery,and the importance of finding the optimal doping ratio.

Delivery of Nucleic Acid Tau-ASOs into Mouse Brain for Gene Knockdown

The efficiency of the mixed lipidoid formulations for ASO delivery invitro by delivering ASO targeting GFP mRNA to HEK cells stablyexpressing green fluorescent protein (GFP) (FIG. 3B) was evaluated. TheNT1-O14B alone showed no GFP silencing effect (10:0 ratio in FIG. 3B),indicating that this lipidoid alone is not effective for delivering ASOintracellularly. However, GFP silencing was observed when the ASO wasdelivered using LNPs containing a mixture of NT1-O14B and 306-O12B-3.When the doping ratio of 306-O12B-3 was greater than 50% (i.e. 5:5weight ratio or more in favor of 306-O12B-3), the GFP silencing inGFP-HEK cells was observed, silencing efficiency increasing as the306-O12B-3 doping ratio increased. Scrambled ASO delivered byLipofetamine 2000 (LPF 2K) showed no GFP silencing, indicating the GFPsilencing was truly ASO sequence-specific.

Whether the mixed lipidoid formulation (NT1-O14B and 306-O12B-3) coulddeliver ASO to the brain and mediate the gene knockdown in vivo was thenexplored. Tau was chosen as a therapeutic target and designed ASOtargeting tau mRNA, since ASO-mediated tau reduction has shown promisingresults in the treatment of Alzheimer's disease (AD) after the localinjection of the Tau-ASO using an intracerebroventricular (ICV)pump.^(31, 32)

The sequence of Tau-ASO was chosen according to the publishedliterature³¹ and was synthesized by IDT. Tau-ASO was supplied containingchemical modification with phosphorothioate groups between each nucleicacid and 2′-O-methoxyethyl in the 5 nucleotides on the 5′- and3′-termini of ribose to improve efficacy. To formulate the ASO forintravenous injection, the ASO with the formulated LNP solution is mixedat a weight ratio of 1/15 (ASO to total lipids). Each mouse receivedfive injections of 1 mg/kg ASO, with each injection spaced three daysapart. The mice were sacrificed four days after the last injection,perfused and the brain tissues were harvested and homogenized to extractthe total RNA. The total tau mRNA levels were analyzed by quantitativePCR. As shown in FIG. 3C, when ASO was delivered using either pureNT1-O14B or pure 306-O12B-3, no tau mRNA reduction in the brain tissueswas detected. For the mixed lipidoid formulations, only NT1-O14B and306-O12-3 with a w/w ratio of 5:5 and 3:7 displayed tau mRNA reductionin the brain. These two formulations resulted in ˜25% and ˜50% mRNAreduction, respectively. No tau mRNA silencing was observed in the mixedlipidoid formulations in other ratios (i.e. 7:3 and 1:9).

To confirm the ASO delivery resulted in functional knockdown of tau, wealso checked the tau protein level of the ASO treated mice using ELISA(FIG. 3D). Comparing with the untreated group, mice treated with Tau-ASOformulated in NT1-O14B/306-O12B-3 (3:7 w/w) showed substantially reducedtotal tau protein level. Furthermore, scrambled Tau-ASO was deliveredwith the best-performing ratios (NT1-O14B/306-O12B-3 at 3:7 w/w) usingthe exact same method as that of functional ASO. As shown, no tau mRNAsilencing effect nor tau protein reduction was detected, demonstratingthe tau knockdown is specifically due to sequence-specific ASOsilencing.

Delivery of GFP-Cre Fusion Protein for Gene Recombination in the Ai14Mouse Brain

GFP fused Cre recombinase was chosen as a model protein for the study,using the Ai14 model mouse line (FIG. 4A). The Ai14 mouse line containsa flox-stop-flox tdTomato construct. The successful intracellulardelivery of Cre protein into the cells of Ai14 mouse leads to the generecombination and turns on the tdTomato expression which can be directlyvisualized as red fluorescence signal without additional staining. Here,(−27)GFP-Cre protein was used. NT1-O14B LNPs doped with PBA-Q76-O16B waschosen, as these nanoparticles could successfully deliver (−27)GFP-Cre.The weight ratio of NT1-O14B and PBA-Q76-O16B was fixed at 3:7, based onthe results observed from the AmB and ASO delivery. Lipid formulationswere prepared using the approaches described for the formulation for ASOdelivery. Briefly, the (−27)GFP-Cre protein was mixed with LNPs at aweight ratio of 1/4 and incubated the solution for 15 min at roomtemperature before intravenous injection. Mice were injected four timeswith a dose of 50 μg protein per injection. Five days after the lastinjection, the mice were sacrificed and brain tissues were collected,fixed, and dehydrated. Then the tissues were cryo-sectioned into 15 μmslices and counter-stained with DAPI for fluorescence imaging. As shownin FIG. 4B, strong tdTomato signals were observed in multiple regions ofthe brain, including cerebral cortex, hippocampus and cerebellum. Incontrast, no tdTomato expression in the brain was observed for the miceinjected with LNP formulations using either pure NT1-O14B (10:0) or purePBA-Q76-O16B (0:10).

Delivery of Different Formulation of GFP-Cre Fusion Protein for GeneRecombination in the Ai14 Mouse Brain

NT1-O14B was doped into various lipid nanoparticle formulations,including 306-O12B, PBA-Q76O16B, Dlin-MC3, to investigate the doped LNPformulation for Cre mRNA delivery into the brain of Ai14 mouse throughi.v. injection. The weight ratio of NT1-O14B and the other ionizablelipid (e.g. 306-O12B, PBA-Q76O16B, Dlin-MC3) is 3:7. To formulate stableLNP, other co-lipids including(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG2000)), cholesterol and DOPE were also included.mRNA encoding Cre recombinase was loaded into the LNP, and injected inAi14 mice. The mice were sacrificed at a specific time point, and braintissues were collected, fixed, and dehydrated. Then the tissues werecryo-sectioned into 15 μm slices and counter-stained with DAPI forfluorescence imaging. The tdTomato signals were observed in multipleregions of the brain, indicating the successful delivery of Cre mRNAinto brain cells with such LNP formulation through systemic injection.The fluorescence images of section of Ai14 mouse brain were shows inFIGS. 19A-19N, FIGS. 20A-20B, and FIGS. 21A-21B. The NT1-O14B doped306-O12B showed highest brain delivery comparing with that doped inPBA-O76O16B or Dlin-MC3 LNP.

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INCORPORATION BY REFERENCE

All U.S. and PCT patent publications and U.S. patents mentioned hereinare hereby incorporated by reference in their entirety as if eachindividual patent publication or patent was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

OTHER EMBODIMENTS

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

1-49. (canceled)
 50. A compound of formula I: Y—W—R^(Lipid)   (I), or a pharmaceutically acceptable salt thereof, wherein Y is

W is —NR²⁰—, —O—, or —S—; and R²⁰ is R^(Lipid), H, C₁₋₆ alkyl, C₂₋₆ alkenyl, or C₂₋₆ alkynyl; and each instance of R^(Lipid) independently of the structure: wherein:

each instance of R¹ and R²is independently —H, —OH, —NHR³⁰, or SH; R³ and R⁴ are both —H; or R³ and R⁴ are taken together to form an oxo (═O) group; Z is —CH₂—, —O—, NR³⁰—, or —S—; X and Y are independently —CH₂—, —NR³⁰—, —O—, —S—, or —Sc—; m is an integer selected from 1-3; n is an integer selected from 1-14; p is 0 or 1; q is an integer selected from 1-10; t is 0 or 1; and R³⁰ is —H, C₂₋₆ alkenyl, or C₂₋₆ alkynyl.
 51. The compound of claim 50, wherein, in at least one instance of R^(Lipid), R³ and R⁴ are taken together to form an oxo (═O) group.
 52. The compound of claim 50, wherein, in at least one instance of R^(Lipid), each instance of R¹ and R² is independently —H or —OH.
 53. The compound of claim 52, wherein, in at least one instance of R^(Lipid), the R¹ and R² are —H.
 54. The compound of claim 50, wherein, in at least one instance of R^(Lipid), Z is —CH₂—, —O—, or —NR³⁰—.
 55. The compound of claim 54, wherein, in at least one instance of R^(Lipid), R¹ and R² are —H, R³ and R⁴ are taken together to form an oxo (═O) group, and Z is O.
 56. The compound of claim 50, wherein, in at least one instance of R^(Lipid), R¹ is —H, R² is —OH, R³ and R⁴ are —H, and Z is —CH₂—.
 57. The compound of claim 50, wherein, in at least one instance of R^(Lipid), X and Y are independently —CH₂— or —O—.
 58. The compound of claim 57, wherein, in at least one instance of R^(Lipid), X and Y are both —CH₂—.
 59. The compound of claim 50, wherein, in at least one instance of R^(Lipid), m is 1 or
 2. 60. The compound of claim 50, wherein, in at least one instance of R^(Lipid), n is an integer selected from 4-12.
 61. The compound of claim 50, wherein, in at least one instance of R^(Lipid), p is
 1. 62. The compound of claim 50, wherein, in at least one instance of R^(Lipid), q is an integer selected from 2-8.
 63. The compound of claim 50, wherein, in at least one instance of R^(Lipid), t is
 0. 64. The compound of claim 50, wherein, in at least one instance of R^(Lipid), t is
 1. 65. The compound of claim 50, wherein the compound is selected from the group consisting of:


66. A lipidoid composition, comprising a plurality of compounds of claim
 50. 67. The lipidoid nanoparticle of claim 66, wherein the lipidoid nanoparticle has a particle size of 25 nm to about 1000 nm.
 68. The lipidoid composition of claim 66, further comprising a protein.
 69. The lipidoid composition of claim 66, further comprising a small molecule
 70. The lipidoid composition of claim 66, further comprising a nucleic acid.
 71. A pharmaceutical composition, comprising a lipidoid nanoparticle of claim 66; and a pharmaceutically acceptable carrier or excipient. 